There is great interest in the study of biologically active oligosaccharides, mainly because of the appreciation of their potential in biology and medicine. This interest has generated a growing need for methods of efficiently and inexpensively preparing oligosaccharides, particularly for the purpose of studying their biochemical function and assessing their potential in therapeutics or as diagnostic tools. Biologically important oligosaccharides are often difficult to obtain from natural sources in sufficient quantities for any systematic biochemical studies. Even when available from natural sources, it is still important to have independently synthesized oligosaccharides to confirm the structure-activity relationships of the molecules.
Until recently, "glycobiologists" have relied on traditional organic chemical synthesis which remains extremely time consuming, cumbersome, and sometimes prohibitively expensive. The formation of isomeric mixtures in chemical glycosylation reactions, the requirements of multiple protection and deprotection steps, and the tedious task of purification of products have been responsible for their vanishing yields. As an alternative to organic chemical synthesis, the enzymatic synthesis of carbohydrates is a particularly attractive approach because the use of enzymes allows stereospecific synthesis and overcomes some of the other limitations of purely chemical syntheses. Thus the difficult process of oligosaccharide synthesis has been aided by enzymatic catalysis and the combined chemo-enzymatic approach has been increasingly reported in the literature, for example, C. A. Compston, C. Condon, H. R Hanna and M. A. Mazid, Carbohydr. Res., 239: 167-176 (1993). Another recent paper describes an electrophoresis-based assay for glycosyltransferase activity which utilizes fluorophore-labelled carbohydrate substrates K. B. Lee, U. R. Desai, M. M. Palcic, O. Hindsgaul and R. J. Linhardt, Anal. Biochem., 205: 108-114 (1992); however, this method also appears time-consuming, tedious or cumbersome in terms of multiple purification steps and ultimate characterization or quantitation of products by sophisticated analytical techniques such as FAB-MS, NMR and capillary zone electrophoresis. A method of carbohydrate synthesis that would permit the convenient separation of reaction products from enzyme and substrates would be a significant advance over currently available methods of carbohydrate synthesis.
The enzymatically-assisted in vitro synthesis of specific oligosaccharides employs three general strategies. These include the use of glycosyltransferases of the Leloir pathway which require sugar-nucleotides as donors, F. Leloir, Science 172: 1299-1303 (1971); H. Nikaido and W. Z. Hassid, Adv. Carbohvdr. Chem. Biochem., 26: 351-483 (1971), the use of non-Leloir pathway enzymes which require sugar-1-phosphate as donors, and the use of glycosidase or glycosylhydrolase-catalyzed reaction for the formation of glycosidic bonds in a kinetic or thermodynamic approach I. Toone, E. S. Simon, M. D. Bednarski and G. M. Whitesides, Tetrahedron, 45: 5365-5422 (1989); S. David, C. Auge and C. Gautheron, Adv. Carbohydr. Chem. Biochem., 49: 175-237 (1991). Several in situ regeneration systems have been reported which avoid the separate tedious preparation of sugar-nucleotides and stoichiometric use of nucleoside mono- and diphosphates that are known inhibitors for the corresponding glycosyltransferases. However, these approaches are generally aimed at the preparative synthesis of oligosaccharides which involve longer purification steps and additional complexities.
The range of carbohydrates that can be produced by a combined chemo-enzymatic approach, particularly synthesis of oligosaccharides, is much greater than simply reproducing the natural biosynthetic reactions for which the enzymes are known to exist. Also, as more glycosyltransferases become available, they could be used to produce a diversity of unnatural oligosaccharides that would be useful in the area of glycoprotein or glycolipid remodelling, M. M. Palcic and O. Hindsgaul, Glycobiology, 1:205-209 (1991). Such studies are extremely important not only for understanding the function of natural glycoconjugates, but also for the design and development of carbohydrate-based therapeutics or diagnostics. However, in order to produce the desired oligosaccharide product, not only must the enzyme and substrates involved in the synthesis be readily available, but suitable techniques for rapidly analyzing and purifying the products should also be available. Conventional synthesis techniques may produce desired oligosaccharides in extremely small quantities, sometimes beyond the detection limit of modern-day sophisticated carbohydrate analytical techniques; thus the lengthy purification steps required to obtain a product of the desired purity often cannot be used successfully. Therefore, traditional approaches for the synthesis and analysis of carbohydrates would appear generally unsuitable to meet most needs of the ordinary glycobiologists.
Conventional methods for the identification, characterization, and synthesis of carbohydrates require lengthy chromatographic separations and the use of sophisticated instruments which are often outside the reach of an ordinary glycobiology laboratory. Hence, it is of interest to provide highly sensitive and convenient methods for the utilization of carbohydrate-modifying enzymes in oligosaccharide synthesis. Such highly sensitive and convenient methods would have a number of uses that are difficult, expensive or impossible, to achieve using currently available techniques. These uses include the detection and purification of carbohydrate products having natural or unnatural structures. For the sake of convenience and economy, it is also of interest to provide for the repeated use of enzyme preparations for carbohydrate synthesis and detection. Furthermore, it is of interest to provide methods of assaying for the presence of carbohydrate-modifying enzymes in a sample for analysis.